The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• genom virový * MeSH
• infekce papilomavirem virologie   MeSH
• integrace viru * MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   MeSH
• metylace DNA MeSH
• mutace MeSH
• onkogenní proteiny virové genetika   MeSH
• tonzilární nádory virologie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

• MeSH
• analýza jednotlivých buněk MeSH
• antigeny CD45 imunologie   MeSH
• deoxyribonukleasa I metabolismus   MeSH
• imunofenotypizace metody   MeSH
• kolagenasy metabolismus   MeSH
• lidé MeSH
• lymfocyty fyziologie   MeSH
• monoklonální protilátky metabolismus   MeSH
• myeloidní buňky fyziologie   MeSH
• nádory diagnóza   imunologie   MeSH
• palladium metabolismus   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• taxonomické DNA čárové kódování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The therapy of different advanced-stage malignancies with monoclonal antibodies blocking programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) signaling has had an impressive long-lasting effect in a portion of patients, but in most cases, this therapy was not successful, or a secondary resistance developed. To enhance its efficacy in treated patients, predictive biomarkers are searched for and various combination treatments are intensively investigated. As the downregulation of major histocompatibility complex (MHC) class I molecules is one of the most frequent mechanisms of tumor escape from the host's immunity, it should be considered in PD-1/PD-L1 checkpoint inhibition. The potential for the use of a PD-1/PD-L1 blockade in the treatment of tumors with aberrant MHC class I expression is discussed, and some strategies of combination therapy are suggested.

• MeSH
• antigeny CD274 antagonisté a inhibitory   imunologie   MeSH
• antigeny CD279 antagonisté a inhibitory   imunologie   MeSH
• down regulace * MeSH
• geny MHC třídy I MeSH
• histokompatibilita - antigeny třídy I genetika   MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• monoklonální protilátky terapeutické užití   MeSH
• nádory genetika   imunologie   terapie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH